Our protocol helps scientists to analyze subcellular actomyosin network in individual epithelial cells and also allows them to perform similar analysis at multicellular scale in curved epithelial tissues. Our protocol is a nice example of a Fiji application. It has an intuitive graphical user interface, and it doesn't require any knowledge of scripting.
Therefore, it is highly suitable also for novice users in the field. Although our method was developed for Drosophila egg chambers, it is also applicable outside Drosophila epithelia. We encourage scientists to test our protocol in their model systems.
Use the accompanied test files to get familiar with this protocol. It will help you to decide which part of this protocol is suitable for your individual research question. This part of the protocol allows users to segment epithelial cells in time-lapse movies and subsequently analyze actomyosin pulses in individual cells over time using the Surface Manager.
To begin, open Fiji, and import the macro TissueCellSegmentMovie. ijm as described in the accompanying text protocol. Then, open a bleach-corrected TIFF file, and split the channels of the bleach-corrected file by going to the menu bar, selecting Image, followed by Color, and selecting Split Channels.
Run the uploaded script on the active cell membrane channel of the selected time-lapse movie by pressing the icon Run in the open script. In order to get a nice cell mask, set the Gaussian blur to 2.500 and the cell detection sensitivity to minus one, and click OK.Then, set the estimated noise tolerance between 10 and 20 for time-lapse movies acquired with the 63x objective, and click OK.A generated cell mask appears in the analyzed time-lapse movie, a new window called ParticleStack appears, and a little window called Action Required also appears. From the cell mask on the time-lapse movie, focus only on cells in the center, and select those that can provide complete and well-defined outlines throughout the time-lapse movie.
When the selected cells in the center nicely correspond to the real cell membranes, save ParticleStack as a TIFF file. Open the corresponding ParticleStack TIFF file in Fiji. With the Surface Manager plugin installed in Fiji, open the plugin.
In Surface Manager, click on the Read outline image button, and set the Jaccard index to 60%Loading the outlines may take several minutes depending on the number of cells. Once loaded, each cell will be assigned an S number and appear in the left part of the Surface Manager window. Click on the selected S number so that the imported cell outline from ParticleStack1.
tiff appears on the time-lapse movie. Check each imported S number for cell outline quality throughout the movie, and remove unwanted cells that display incorrect cell outlines. To remove an unwanted cell, highlight the cell outline and click on the Delete button.
Use the Brush tool to correct cell outlines. When finished, save the corrected cells as an RoiSet. zip file.
To identify whether actomyosin pulses are present in the analyzed tissue and to understand the detailed behavior, as well as the directionality of actomyosin signals, begin by opening Fiji. Open a time-lapse movie, load the saved ParticleStack1. tiff cell mask and the bleach-corrected time-lapse movie, then load the Surface Manager plugin and the corresponding region of interest from the RoiSet.
zip file. Next, in Surface Manager, activate a channel with signals of interest. Click the Statistics button to obtain the window called Average gray value Slice by Slice.
The mean and median intensities of the actomyosin signals will be displayed over time in arbitrary units, as well as other parameters related to the cell's area and shape. Save the values as a spreadsheet file by going to the Statistics window, clicking on File, and selecting Save As.Similarly, generate a cell mask, and load it into Surface Manager in order to analyze actomyosin pulses at the tissue scale. This part of the protocol allows users to selectively extract a thin layer of actomyosin in the curved epithelial tissue over time.
These steps are user-friendly and based on an intuitive graphical interface. Open Fiji, and ensure that the Ellipsoid Surface Projection plugin is installed. Then, open a time-lapse movie, and export it as an XML/HDF5 file by clicking on Plugins, going to BigDataViewer, and selecting Export Current Image as XML/HDF5 file.
Next, open the exported file in Ellipsoid Surface Projection by going to Plugins, clicking on BigDataViewer, followed by Ellipsoid Surface Projection, and selecting XML file. This will open a new window with sagittal views of the egg chamber, and a dialog to guide the user through processing will appear. The dialog window, called Ovaries Projection, has several tabs.
In the first dialog tab, called bounding box, define the x and y borders of an egg chamber in the time-lapse movie together with the z width of the bounding box, and press set. The selected parts of the egg chamber are highlighted in pink. Next, define the size of signal blobs in the dialog tab called find blobs in the Ovaries Projection window.
Define the sigma and the minimal peak value. Then, press compute to identify the actomyosin signals, which will appear as green blobs. Retry this step with different parameters until around 100 spots are found.
Identifying the actomyosin signals is a crucial step. It depends on the signal quality and the correct size of the detected blobs. If there are no visible green spots in the image, then the next step will not work.
To design the ellipsoid, continue with the tab called fit ellipsoid. Set random samples to 10, 000, the outside/inside cutoff distance to one to 10, and then click compute. After this, the tab called projection will automatically open and allows for the definition of the surface extraction.
In the projection tab, set up a minimum and maximum projection distance as the width of the desired ellipsoid. The minimal and maximal projection distance must fit so the pink defined ellipsoid region includes the entire outside layer of the egg chamber. Then, define the slice distance as one.
Make sure that the pink region of the ellipsoid with the defined with on the egg chamber is visible before pressing compute. It helps to evaluate the quality of a new ellipsoid fit. Next, set an output width of the ellipsoid to greater than or equal to 800 and a height that is greater than or equal to 400.
Then, set the duration of the surface extraction, and choose either a spherical or a cylindrical projection. If required, flip Z and align Y.Press compute to obtain a surface extraction for both channels in new windows called image. Adjust the brightness and the contrast of the obtained image windows to be able to see the projected actomyosin signals by clicking on Image, going to Adjust, and selecting Brightness/Contrast.
If the surface extraction looks good, save both channels separately as TIFF files. Additionally, save the Log window file for future reference as to how the surface of this particular egg chamber was extracted. Then, merge the channels with a preferred color code in Fiji, and save the results as TIFF files.
This protocol enables scientists to investigate the behavior of actomyosin networks in epithelial tissues. This is only possible when a detailed analysis of actomyosin behavior at the local cellular scale is combined with a similar analysis at the tissue scale. Shown here are representative examples of dynamic myosin II behavior at the local cellular scale and at the tissue scale for control and fat2 mutant Drosophila egg chambers.
On the basal single plane, the green modified regulatory light chain of myosin II signals move perpendicular to the anterior-posterior axis of control egg chambers. This polarity is lost in fat2 mutant egg chambers and leads to anisotropic myosin II pulses, oscillations At the tissue level, the green modified regulatory light chain of myosin II in the control sample generates the synchronized force required to promote epithelial rotation. In contrast, the follicle epithelium of a fat2 mutant egg chamber pulse strongly at the basal side and fails to generate the synchronized force required to promote epithelial rotation.
In a thin apical region of the Drosophila egg chamber, the fat2 mutant also presents with altered dynamic behavior of the green modified regulatory light chain of myosin II.After watching this video, you should have a good idea of how to analyze a actomyosin network at local and tissue scale in Drosophila egg chambers using Fiji. For further practical tips and troubleshooting, please see the discussion part after the protocol. If you have any questions related to Fiji or our plugins, see the respective webpages or contact us.
